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Yanhua Liu, Dongjuan Yuan, Zexin Zhao, Dongming Lan, Bo Yang, Yonghua Wang
Penicillium camembertii (PCL), a mono- and di-acylglycerol lipase (DGL), has the vital potential in the oil chemistry for food industry. However, known DGLs are mesophilic enzymes which restricts its application in the industry. To improve thermostability of PCL, we used amino acid substitution by comparison of amino acids compositions of PCL and protein sequences from typical thermophilic bacteria. Then, some conservative residues around active center were avoided to mutate according to homologous alignment analyses...
April 18, 2018: Biochemical and Biophysical Research Communications
Stephan T Kudlacek, Lakshmanane Premkumar, Stefan W Metz, Ashutosh Tripathy, Andrey A Bobkov, Alexander Matthew Payne, Stephen Graham, James A Brackbill, Michael J Miley, Aravinda M de Silva, Brian Kuhlman
The spread of dengue (DENV) and Zika virus (ZIKV) is a major public health concern. The primary target of antibodies (Abs) that neutralize DENV and ZIKV is the envelope (E) glycoprotein, and there is interest in using soluble recombinant E (sRecE) proteins as subunit vaccines. However, the most potent neutralizing antibodies against DENV and ZIKV recognize epitopes on the virion surface that span two or more E proteins. Therefore, to create effective DENV and ZIKV vaccines, presentation of these quaternary epitopes may be necessary...
April 20, 2018: Journal of Biological Chemistry
Bijender Singh, K K Sharma, Amit Kumari, Anil Kumar, S K Gakhar
A thermostable and protease-resistant HAP-phytase of Sporotrichum thermophile was over-expressed in Pichia pastoris ×33. Purified recombinant phytase displayed all its biochemical properties similar to wild type. Molecular modeling and docking of phytase with various substrates showed differential binding patterns with GoldScore values ranging from 40.61 to 79.78. Docking with different substrates revealed strong binding affinity with ATP and phytic acid, while the lowest with AMP and phosphoenol pyruvate...
April 17, 2018: International Journal of Biological Macromolecules
Zi-Yue Wang, Li-Juan Wang, Qianyi Zhang, Bo Tang, Chun-Yang Zhang
DNA methylation is an important epigenetic modification in human genomes. Herein, we develop a single quantum dot (QD)-based nanosensor for sensitive detection of DNA methylation at both CpG and non-CpG sites using tricyclic ligation chain reaction (LCR)-mediated QD-based fluorescence resonance energy transfer (FRET). We design two sets of DNA probes (X and Y, X' and Y') for methylated DNA assay. In the presence of thermostable DNA ligase, probes X and Y may adjacently hybridize with the methylated DNA to obtain the ligated XY products which may function as the templates for probes X' and Y' to generate the X'Y' products...
February 7, 2018: Chemical Science
Qiang Zhang, Vladimir J N Bykov, Klas G Wiman, Joanna Zawacka-Pankau
The TP53 tumor suppressor gene is frequently inactivated in human tumors by missense mutations in the DNA binding domain. TP53 mutations lead to protein unfolding, decreased thermostability and loss of DNA binding and transcription factor function. Pharmacological targeting of mutant p53 to restore its tumor suppressor function is a promising strategy for cancer therapy. The mutant p53 reactivating compound APR-246 (PRIMA-1Met ) has been successfully tested in a phase I/IIa clinical trial. APR-246 is converted to the reactive electrophile methylene quinuclidinone (MQ), which binds covalently to p53 core domain...
April 18, 2018: Cell Death & Disease
Ahmad Firdaus B Lajis
For decades, microbial lipases are notably used as biocatalysts and efficiently catalyze various processes in many important industries. Biocatalysts are less corrosive to industrial equipment and due to their substrate specificity and regioselectivity they produced less harmful waste which promotes environmental sustainability. At present, thermostable and alkaline tolerant lipases have gained enormous interest as biocatalyst due to their stability and robustness under high temperature and alkaline environment operation...
2018: Journal of Lipids
Estrella Duque, Abdelali Daddaoua, Baldo F Cordero, Zulema Udaondo, Carlos Molina-Santiago, Amalia Roca, Jennifer Solano, Eduarda Molina-Alcaide, Ana Segura, Juan-Luis Ramos
The success of second-generation (2G) ethanol technology relies on the efficient transformation of hemicellulose into monosaccharides and, particularly, on the full conversion of xylans into xylose for over 18% of fermentable sugars. We sought new hemicellulases using ruminal liquid, after enrichment of microbes with industrial lignocellulosic substrates and preparation of metagenomic libraries. Among 150 000 fosmid clones tested, we identified 22 clones with endoxylanase activity and 125 with β-xylosidase activity...
April 17, 2018: Microbial Biotechnology
Li-Qiang Fan, Ming-Wei Li, Yong-Jun Qiu, Qi-Ming Chen, Si-Jing Jiang, Yu-Jie Shang, Li-Ming Zhao
Gamma-amino butyric acid (GABA) is an important bio-product used in pharmaceuticals, functional foods, and a precursor of the biodegradable plastic polyamide 4 (Nylon 4). Glutamate decarboxylase B (GadB) from Escherichia. coli is a highly active biocatalyst that can convert L-glutamate to GABA. However, its practical application is limited by the poor thermostability and only active under acidic conditions of GadB. In this study, we performed site-directed saturation mutagenesis of the N-terminal residues of GadB from Escherichia coli to improve its thermostability...
April 13, 2018: Journal of Biotechnology
Jingxu Guo, Alun R Coker, Steve P Wood, Jonathan B Cooper, Ronan M Keegan, Nasir Ahmad, Majida Atta Muhammad, Naeem Rashid, Muhummad Akhtar
Pullulan-hydrolysing enzymes, more commonly known as debranching enzymes for starch and other polysaccharides, are of great interest and have been widely used in the starch-saccharification industry. Type III pullulan hydrolase from Thermococcus kodakarensis (TK-PUL) possesses both pullulanase and α-amylase activities. Until now, only two enzymes in this class, which are capable of hydrolysing both α-1,4- and α-1,6-glycosidic bonds in pullulan to produce a mixture of maltose, panose and maltotriose, have been described...
April 1, 2018: Acta Crystallographica. Section D, Structural Biology
Xiuting Li, Christopher Batchelor-McAuley, Javor K Novev, Richard G Compton
An optimised thermostated electrochemical cell is designed and implemented. This is informed by experimental and computational studies characterizing the extent to which the thermostating of an electrochemical cell via a heated bath can be realised, both with the cell closed and open to the environment. The heat transfer in the system is simulated and probed experimentally; special emphasis is put on heat loss due to radiation and evaporation. Experiments and simulations demonstrate that these two mechanisms of heat transfer lead to a steady temperature in the cell that differs from that of the thermostat by ∼0...
April 13, 2018: Physical Chemistry Chemical Physics: PCCP
Christina E Boville, David K Romney, Patrick James Almhjell, Michaela Sieben, Frances H Arnold
The use of enzymes has become increasingly widespread in synthesis as chemists strive to reduce their reliance on organic solvents in favor of more environmentally benign aqueous media. With this in mind, we previously endeavored to engineer the tryptophan synthase β-subunit (TrpB) for production of noncanonical amino acids that had previously been synthesized through multi-step routes involving water-sensitive reagents. This enzymatic platform proved effective for the synthesis of analogs of the amino acid tryptophan (Trp), which are frequently used in pharmaceutical synthesis as well as chemical biology...
April 13, 2018: Journal of Organic Chemistry
Mohamed Amine Gomri, Agustín Rico-Díaz, Juan-José Escuder-Rodríguez, Tedj El Moulouk Khaldi, María-Isabel González-Siso, Karima Kharroub
Proteases have numerous biotechnological applications and the bioprospection for newly-thermostable proteases from the great biodiversity of thermophilic microorganisms inhabiting hot environments, such as geothermal sources, aims to discover more effective enzymes for processes at higher temperatures. We report in this paper the production and the characterization of a purified acid protease from strain OA30, a moderate thermophilic bacterium isolated from an Algerian hot spring. Phenotypic and genotypic study of strain OA30 was followed by the production of the extracellular protease in a physiologically-optimized medium...
April 12, 2018: Microorganisms
Yoshinori Matsuura, Michiyo Takehira, George I Makhatadze, Yasumasa Joti, Hisashi Naitow, Naoki Kunishima, Katsuhide Yutani
In order to elucidate the contribution of charged residues to protein stabilization at temperatures of over 100 °C, we constructed many mutants of the CutA1 protein ( EcCutA1) from Escherichia coli. The goal was to see if one can achieve the same stability as for a CutA1 from hyperthermophile Pyrococcus horikoshii that has the denaturation temperature near 150 °C. The hydrophobic mutant of EcCutA1 ( Ec0VV) with denaturation temperature (Td) of 113.2 °C was used as a template for mutations. The highest Td of Ec0VV mutants substituted by a single charged residue was 117...
April 12, 2018: Biochemistry
Peng Wang, Yatao Wu, Jie Liu, Ping Song, Shan Li, Xinxin Zhou, Guoping Zhu
Monomeric isocitrate dehydrogenases (IDHs) have a single polypeptide sizing around 85 kDa. The IDH2 from the opportunistic bacterium Acinetobacter baumannii (AbIDH2) with a mass of 83 kDa was formerly recognized as a typical monomeric IDH. However, both size exclusion chromatography and analytical ultracentrifugation analysis indicated that AbIDH2 exists as a homodimer in solution. The crystallographic study of the substrate/coenzyme-free AbIDH2 gave a dimeric structure and each subunit contained a domain I and a domain II...
April 10, 2018: International Journal of Molecular Sciences
John N Milligan, Raghav Shroff, Daniel J Garry, Andrew D Ellington
Strand-displacing polymerases are a crucial component of isothermal amplification (IA) reactions where the lack of thermal cycling reduces equipment needs and improves time-to-answer, especially for point of care applications. In order to improve the function of strand displacing polymerases, we have developed an emulsion-based directed evolution scheme, high temperature isothermal compartmentalized self-replication (HTI-CSR) that does not rely on thermal cycling. Starting from an algorithm-optimized shuffled library of exonuclease-deficient Family A polymerases from Geobacillus stearothermophilus (Bst LF) and Thermus aquaticus (Klentaq), we have applied HTI-CSR to generate a more thermostable strand displacing polymerase variant that performs well in Loop-mediated Isothermal Amplification (LAMP) and Rolling Circle Amplification (RCA), even after thermal challenges of up to 95°C that lead to better primer annealing...
April 9, 2018: Biochemistry
Krishnendu Pramanik, Sunayana Saren, Soumik Mitra, Pallab Kumar Ghosh, Tushar Kanti Maiti
Lipase (triacylglycerol acylhydrolase, EC catalyzes tri-, di-, and monoacyl glycerol of fat into glycerol and fatty acids. It has important roles in the digestion of lipids in living organisms and industrially as laundry detergents along with proteases. The microbial lipases are more stable, active and economically feasible compared to plant and animal sources. Hence, much attention was given to the maximum production of the enzyme from the microbial sources. The phylogenetic analysis revealed that the amino acid sequence of lipase protein and their corresponding cDNA of Pseudomonas aeruginosa clustered with Pseudomonas stutzeri among different species of Pseudomonas, while P...
March 19, 2018: Computational Biology and Chemistry
Sergio F Bodini, Marzio Malizia, Annalisa Tortelli, Luca Sanfilippo, Xingpeng Zhou, Roberta Arosio, Marzia Bernasconi, Stefano Di Lucia, Angela Manenti, Pompeo Moscetta
A novel tool, the DAMTA analyzer (Device for Analytical Monitoring and Toxicity Assessment), designed for fully automated toxicity measurements based on luminescent bacteria as well as for concomitant determination of chemical parameters, was developed and field-tested. The instrument is a robotic water analyzer equipped with a luminometer and a spectrophotometer, integrated on a thermostated reaction plate which contains a movable carousel with 80 cuvettes. Acute toxicity is measured on-line using a wild type Photobacterium phosphoreum strain with measurable bioluminescence and unaltered sensitivity to toxicants lasting up to ten days...
April 5, 2018: Ecotoxicology and Environmental Safety
Dong-Xu Jia, Teng Wang, Zi-Jian Liu, Li-Qun Jin, Jia-Jia Li, Cheng-Jun Liao, De-Shui Chen, Yu-Guo Zheng
Glucose isomerase (GI) responsible for catalyzing the isomerization from d-glucose to d-fructose, was an important enzyme for producing high fructose corn syrup (HFCS). In a quest to prepare HFCS at elevated temperature and facilitate enzymatic recovery, an effective procedure for whole cell immobilization of refractory Thermus oshimai glucose isomerase (ToGI) onto Celite 545 using tris(hydroxymethyl)phosphine (THP) as crosslinker was established. The immobilized biocatalyst showed an activity of approximate 127...
April 4, 2018: Journal of Bioscience and Bioengineering
D Simón, A M Borreguero, A de Lucas, J F Rodríguez
The recycling of any kind of plastic to convert it in valuable products is one of the main challenges of today's society. Besides, if the recycling process is itself green, then it would be a great achievement. This paper reviews the way covered from the first attempts of reusing the polyurethane (PU) scraps as a filler for cushions to the last chemical routes employing green recycling agents. Polyurethane is the 6th most used polymer all over the world with a production of 18 millions tons per year, which means a daily production of PU specialties greater than 1 million of cubic meters, equivalent to the volume of the Empire State Building...
April 3, 2018: Waste Management
A A Tyurin, K V Kabardaeva, O N Mustafaev, O S Pavlenko, N S Sadovskaya, V S Fadeev, E A Zvonova, I V Goldenkova-Pavlova
A recombinant DNA in which the interferon αA (IFN-αA) gene sequence is integrated into a loop region of the gene coding thermostable lichenase was constructed. This approach of insertion fusion with thermostable lichenase is advantageous in terms of increasing the solubility, stability, and production of the fusion partner in soluble form in general and in the periplasm of bacterial cells in particular. Thus, the insertion of IFN-αA into the loop (53 a.a.) of thermostable lichenase from Clostridium thermocellum resulted in effective expression of the soluble form of the recombinant protein in the periplasm of Escherichia coli without any compromise in biological activity of IFN-αA, while the thermostable lichenase retained its ability for functional folding without dramatic loss of its basic activity and thermostability...
March 2018: Biochemistry. Biokhimii︠a︡
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